Interactions between an anti-sigma protein and two sigma factors that regulate the pyoverdine signaling pathway in Pseudomonas aeruginosa

Abstract

Background: Synthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity.

Results: To investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.

Conclusions: These data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.

Figure 1

Co-purification of PvdS with FpvR _1–89 –TAP from P. aeruginosa . Soluble protein was prepared from P. aeruginosa PAO1 fpvR expressing plasmid-borne (pUCP23) or chromosomally-integrated (ctx) FpvR_1–89 fused to a C-terminal TAP tag. Protein was purified using the TAP protocol and the purified protein analyzed by Western blotting for FpvR_1–89-CBP or PvdS. (A) anti-CBP; (B) anti-FpvR; (C) anti-PvdS. A mock purification was carried out with P. aeruginosa PAO1 fpvR carrying pUCP23 without the fpvR _1–89-TAP fusion as a negative control for the TAP tag purification procedure. The positions of molecular weight markers are shown.

Figure 2

The activity of PvdS and FpvI in the presence and absence of FpvR _1–89 . β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing either (A) pMP190::P_fpvA_lacZ or (B) pMP190::P_pvdE_lacZ, along with pETDuet expressing FpvR_1–89 and either (A) FpvI or (B) PvdS. An empty pETDuet control is also shown to control for background expression of lacZ from pMP190::P_fpvA_lacZ or pMP190::P_pvdE_lacZ. Averages were obtained from three biological replicates. Error bars are ±1 SD.

Figure 3

Co-purification of FpvR _1–89 with either His ₆ -FpvI or His ₆ -PvdS from E. coli . Soluble protein was obtained from E. coli MC1061 (DE3) co-expressing either His₆-FpvI and FpvR_1-89 or His₆-PvdS and FpvR_1–89. Protein was purified by nickel affinity chromatography via the His₆-tags on PvdS and FpvI and analyzed by SDS-PAGE (top panels) and Western blotting (lower panels) using anti-FpvI, anti-PvdS or anti-FpvR antibodies as shown. (A) Co-purification of FpvR_1–89 with His₆-FpvI; (B) co-purification of FpvR_1-89 with His₆-PvdS. The positions of molecular weight markers are shown.

Figure 4

The activity of FpvI and PvdS mutants in the presence of FpvR _1–89 . β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::P_fpvA_lacZ or (B) pMP190::P_pvdE_lacZ, along with pETDuet expressing FpvR_1–89 and (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Mutants that were further investigated in Figure 5 are indicated in bold. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.

Figure 5

The activity of FpvI and PvdS mutants in the absence of FpvR _1–89 . β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing (A) pMP190::P_fpvA_lacZ or (B) pMP190::P_pvdE_lacZ, along with pETDuet expressing (A) mutant FpvI or (B) mutant PvdS. Dark grey bars: FpvI and PvdS mutants generated by error prone PCR. Patterned bars: FpvI and PvdS mutants engineered by site directed mutagenesis. Light grey bars: WT FpvI or PvdS. Data were obtained from three biological replicates and error bars are ±1 SD. Statistically significant difference to WT according to Student's T-test is indicated: *p <0.05, **p <0.01.

Figure 6

The location of mutations in FpvI and PvdS that increased sigma factor activity in the presence of FpvR _1–89 . The approximate location of mutations are shown on the ECF sigma factor functional regions two (2.1-2.4) and four (4.1 and 4.2) of (A) FpvI and (B) PvdS according to the following classifications: ▲mutation(s) that gave higher activity in the presence, not absence of FpvR_1–89; ■ mutation(s) that gave higher activity whether or not FpvR_1–89 was present; ● mutation(s) that gave higher activity in the presence of FpvR_1–89 and were not tested in the absence of FpvR_1–89. Each mutant is represented on a different line.

Figure 7

The activity of FpvI and PvdS C-terminal deletion mutants. (A) An alignment of FpvI showing the 5 amino acid C-terminal deletion and PvdS showing the 13 amino acid C-terminal deletion. (B and C) β-galactosidase assays were carried out with E. coli MC1061 (DE3) containing pMP190::P_fpvA_lacZ or pMP190::P_pvdE_lacZ, along with pETDuet expressing FpvR_1–89 and C-terminal deletion mutants of (B) FpvI or (C) PvdS. Values are compared to WT FpvI and PvdS and were obtained from three biological replicates. Error bars are ±1 SD.